The CCL3 family of chemokines and innate immunity cooperate in vivo in the eradication of an established lymphoma xenograft by rituximab

The therapeutic mAb rituximab induced the expression of the CCL3 and CCL4 chemokines in the human lymphoma line BJAB following binding to the CD20 Ag. Induction of CCL3/4 in vitro was specific, was observed in several cell lines and freshly isolated lymphoma samples and also took place at the protein level in vitro and in vivo. To investigate the role of these beta-chemokines in the mechanism of action of rituximab, we synthesized a N-terminally truncated CCL3 molecule CCL3(11-70), which had antagonist activity on chernotaxis mediated by either CCL3 or BJAB supernatant. We also set up an established s.c. BJAB tumor model in athymic mice. Rituximab, given weekly after tumors had reached 250 MM2, led to complete disappearance of the lymphoma within 2-3 wk. Treatment of mice with cobra venom factor showed that complement was required for rituximab therapeutic activity. Treatment of BJAB tumor bearing mice every 2 days with the CCL3(11-70) antagonist, starting 1 wk before rituximab treatment, had no effect on tumor growth by itself, but completely inhibited the therapeutic activity of the Ab;.To determine whether CCL3 acts through recruitment/activation of immune cells, we specifically depleted NK cells, polymorphonuclear cells, and macrophages using mAbs, clodronate treatment, or Rag2(-/-)cy(-/-) mice'. The data demonstrated that these different cell populations are involved in BJAB tumor eradication. We propose that rituximab rapidly activates complement and induces beta-chemokines in vivo, which in turn activate the innate immunity network required for efficient eradication of the bulky BJAB tumor. The Journal of Immunology, 2007,178: 6616-6623. tor mechanisms is a matter of large debate (6). Different animal models of lymphoma have been described that have emphasized a role for either Ab-dependent cytotoxicity by NK cells and neutrophils (7, 8) or for phagocytosis mediated by the monocyte network (9, 10) or by complement activation (11- 13). These mechanisms, however, are likely to be linked to each other. For example the complement cascade may cause direct target cell lysis by the membrane attack complex as well as induce recruitment and activation of different immune effector cells after activation and release of potent anaphylatoxins like C5a and C3a (14, 15). Finally, the role of different mechanisms is likely to vary in different tumor types or localizations (10). Rituximab binding to the CD20 molecule also activates restricted intracellular signaling pathways. Recently we have investigated these direct effects of rituximab on lymphoma cells using a gene chip screening approach (16) and found several genes induced by the Ab in different lymphoma cells. Two of the genes of biological interest were CCL3 (Mipl-alpha) and CCL4 (Mipl-beta), which were up-regulated in BJAB lymphoma cells in vitro after stimulation with rituximab. CCL3 and CCL4 belong to the family of 0 (CC) chemokines expressed generally in a coordinated fashion by a number of cell types, including activated B cells, monocytes, mast.cells, fibroblasts, and epithelial cells (17). They are highly conserved between mouse and human models (18) and both signal through the CCR1 and CCR5 receptors. These receptors are also shared by other beta-chemokine family members such as CCL5 (RANTES). CCL3 and CCL4 induce the infiltration of neutrophils, macrophages, NK cells, and T cells in tumors. CCL3 has been implicated in antitumor activity in several mouse models of solid tumors (19-23). In addition CCL3 has the capacity to activate certain cll types (20, 24), to affect proliferation of hemopoietic cells (25, 26), and has osteoclastic properties in vitro or in vivo